Focal modulation microscopy with image-based aberration correction

Abstract

In biomedical fields, the optical fluorescence microscopy is an attractive imaging technique for non-invasive in vivo studies. Confocal microscopy (CM) has the optical sectioning ability that can image thin sections of a thick tissue with sub-micron spatial resolution. The design for CM to deliver performance close to the diffraction limit is highly optimized. However, the performance of CM is degraded when imaging depth is going deeper from the surface. A biological specimen has a highly scattering media and a variation in refractive index due to its heterogeneous structures from macroscopic to microscopic scales. These two phenomena introduce the detrimental effects on imaging quality A large number of scattering and absorption events restrict the propagation of photons to desired plane in biological sample. Recently a novel technique called FMM (focal modulation microscopy) is developed. FMM improves the penetration depth up to several hundreds of micrometers while maintaining diffraction limited spatial resolution. The excitation beam from continuous laser can generate intensity modulation signal only in focal plane of objective: this is called focal modulation. Focal modulation enables to distinguish desired signal with modulation and out of focus signal with non-modulation. Using the extracted modulation component from detection signal is effective technique to suppress the out-of focus signal. In this paper, the high speed FMM is proposed. For enhancement of modulation speed, beat phenomenon by two AOMs has been used in the field of heterodyne interferometer. Two AOMs driven at slightly different radio frequencies (RF) generate two spatially separated beams with different optical frequencies. They are maintained in parallel non-overlapping manner before reaching the focal plane. In the focal volume, two beams interfere and generate beating signals resulting from the heterodyne interference. Finally the modulation frequency is difference between RF freq...